Methods and device for biomolecule characterization

• US 20030207326A1
• Filed: 05/01/2002
• Published: 11/06/2003
• Est. Priority Date: 05/01/2002
• Status: Active Grant

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Su, Xing, Berlin, Andrew A.
• Granted Patent: US 7,744,816 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

The methods and apparatus 100, disclosed herein are of use for sequencing 150 and/or identifying 160 proteins 230, 310, polypeptides 230, 310 or peptides 230, 310. Proteins 230, 310 containing labeled amino acid residues may be synthesized and passed through nanopores 255, 330. A detector 257, 345 operably coupled to a nanopore 255, 330 may detect labeled amino acid residues as they pass through the nanopore 255, 330. Distance maps 140 for each type of labeled amino acid residue may be compiled. The distance maps 140 may be used to sequence 150 and/or identify 160 the protein 230, 310. In different embodiments of the invention, amino acid residues labeled with luminescent labels 235, 245 or nanoparticles 315 may be detected using photodetectors 257 or electrical detectors 345. Apparatus 100 of use for protein 230, 310 sequencing 150 and/or identification 160 are also disclosed herein.

Claims

1. A method comprising:

a) obtaining one or more labeled proteins, polypeptides or peptides;

b) passing the labeled proteins, polypeptides or peptides through one or more nanopores;

c) detecting labeled amino acid residues in the labeled proteins, polypeptides or peptides;

d) compiling an amino acid distance map for each type of labeled amino acid; and

e) identifying the protein based on the distance maps.

2. The method of claim 1, further comprising:

a) placing a template nucleic acid into at least one chamber, each chamber to contain a different labeled amino acid; and

b) producing one or more labeled proteins, polypeptides or peptides encoded by the template nucleic acid.

3. The method of claim 1, further comprising:

a) obtaining one or more proteins, polypeptides or peptides from a biological sample; and

b) labeling the proteins, polypeptides or peptides post-translationally.

4. The method of claim 1, wherein the protein, polypeptide or peptide is identified by comparing the distance maps with a library of amino acid distance maps.

5. The method of claim 2, wherein each chamber is operably coupled to a different set of nanopores.

6. The method of claim 1, wherein each nanopore is operably coupled to a detector.

7. The method of claim 1, wherein only one labeled protein, polypeptide or peptide passes through a nanopore at a time.

8. The method of claim 2, wherein the labeled amino acids in each chamber represent between about 0.5% and about 50% of the total amount of the same amino acid in that chamber.

9. The method of claim 1, wherein the length of time between passage of a first labeled amino acid through the nanopore and passage of a second labeled amino acid through the nanopore corresponds to the distance along the labeled protein, polypeptide or peptide between the first and second amino acids.

10. The method of claim 1, wherein the labels are selected from the group consisting of luminescent labels, fluorescent labels, phosphorescent labels, chemiluminescent labels, conductive labels, nuclear magnetic resonance labels, mass spectroscopy labels, electron spin resonance labels, electron paramagnetic resonance labels and Raman labels.

11. The method of claim 1, wherein at least one end of the labeled protein, polypeptide or peptide is attached to an identifiable label.

12. The method of claim 1, wherein the N-terminal end of the labeled protein, polypeptide or peptide is attached to an identifiable label.

13. The method of claim 6, wherein said labeled protein, polypeptide or peptide comprises luminescent labels and said labeled amino acids are detected with a photodetector.

14. The method of claim 6, wherein said labeled protein, polypeptide or peptide comprises nanoparticles and said labeled amino acids are detected with an electrical detector.

15. The method of claim 2, further comprising analyzing a multiplicity of labeled proteins, polypeptides or peptides from each chamber.

16. The method of claim 1, further comprising determining the sequence of the protein, polypeptide or peptide based on the distance maps.

17. An apparatus comprising:

a) at least one sub-device, each sub-device comprising an upper chamber and a lower chamber, said upper and lower chambers separated by sensor layers, the upper and lower chambers of each sub-device in fluid communication through one or more nanopores; and

b) one or more detectors.

18. The apparatus of claim 17, further comprising an electrode in each upper and lower chamber, said electrodes operably coupled to a voltage regulator.

19. The apparatus of claim 17, further comprising a computer operably coupled to the one or more detectors.

20. The apparatus of claim 17, wherein the one or more detectors comprise a photodetector, an electrical detector and/or a voltage detector.

21. The apparatus of claim 17, wherein said sensor layers comprise a support layer, a photon sensing layer and a light opaque layer.

22. The apparatus of claim 21, wherein said photon sensing layer is operably coupled to one or more photodetectors.

23. The apparatus of claim 22, further comprising a light source and an amplifier.

24. The apparatus of claim 17, wherein said sensor layers comprise at least one conducting layer and at least two insulating layers.

25. The apparatus of claim 24, wherein said conducting layer is operably coupled to one or more electrical detectors.

26. The apparatus of claim 17, wherein said nanopore is part of a nanotube or nanochannel.

27. A method comprising:

a) contacting one or more cells with a labeled subunit;

b) obtaining one or more copies of a molecule comprising labeled subunits from the cells;

c) passing the labeled molecule through one or more nanopores;

d) detecting labeled subunits on the labeled molecule;

e) compiling a subunit distance map; and

f) identifying the molecule from the distance map.

28. The method of claim 27, wherein the molecule is selected from the group consisting of a nucleic acid, oligonucleotide, protein, polypeptide, peptide, polysaccharide and lipid.

29. The method of claim 28, wherein the molecule is a protein, polypeptide or peptide and the cells are transformed with an expression vector encoding the protein, polypeptide or peptide.

30. The method of claim 27, further comprising contacting at least two groups of cells with labeled subunits, each group of cells contacted with a different type of labeled subunit.